Exit from mitosis in eukaryotic cells requires the programmed, ubiquitin-mediated degradation of cell cycle regulatory proteins, such as mitotic cyclins and inhibitors of anaphase initiation. These proteins are targeted for degradation by a multisubunit ubiquitin ligase complex known as the cyclosome, or Anaphase Promoting Complex (APC). Dr. Hershko has previously studied the cyclosome and its basic regulation in a cell-free system from clam oocytes that reproduces the early embryonic cell cycles. He now wishes to investigate the molecular mechanisms of the regulation of cyclosome activity in the more complicated human somatic cell cycles. Two WD-40 repeat-containing proteins, called Fizzy (FZY)/Cdc20 and Fizzy-related (FZR)/Cdh1 are required for cyclosome activity in mitosis and in the G1 phase of the cell cycle, respectively. In preliminary experiments he has found that mitotic phosphorylation of the cyclosome from human cells is required for its activation by FZY, but not by FZR. In this project Dr. Hershko plans to investigate the molecular mechanisms by which FZY and FZR stimulate the activity of the cyclosome/APC. He plans to examine the interactions of FZY and FZR with the phosphorylated and dephosphorylated forms of the cyclosome, and to identify subunits involved in these interactions and in substrate binding. Dr. Hershko will also investigate the mechanisms by which the mitotic checkpoint system 'regulates the FZY-activated form of the cyclosome, by biochemical reconstitution approach using purified components. The biochemical analysis of the mechanisms of the regulation of this ubiquitin ligase complex, essential for exit from mitosis, may yield significant insights into the control of division of human cells and into its aberrations in malignant transformation.